Pathogenicity of clade 2.3.2.1 H5N1 highly pathogenic avian influenza virus in American kestrel (Falco sparverius).

Birds of prey, including endangered species, have been infected with H5 highly pathogenic avian influenza viruses (HPAIVs) in several countries. In this present study, we assessed the pathogenicity of the clade 2.3.2.1 H5N1 HPAIV in American kestrels (Falco sparverius) with a view to preventing future outbreaks in raptors. The kestrels were intranasally inoculated with the virus or fed the meat of chicks that had died from viral infection. Kestrels in both groups initially had reduced food intake, showed clinical signs such as depression and neurologic manifestations, and succumbed to the infection within 6 days. The kestrels primarily shed the virus orally from 1 day post-inoculation until death, with an average titre of 104.5-5.7 EID50/ml, which is comparable to the inoculum titre. The viruses replicated in almost all tested tissues; notably, the feather calamuses also contained infectious virions and/or viral genes. Pancreatic lesions were present in several infected birds, as shown in previous cases of HPAIV infection in raptors. These results indicate that kestrels are highly susceptible to infection by clade 2.3.2.1 H5 HPAIVs, which readily occurs through the consumption of infected bird carcasses. Early detection and removal of HPAIV infected carcasses in the field is essential for preventing outbreaks in raptors. RESEARCH HIGHLIGHTS Clade 2.3.2.1 H5 HPAIV caused lethal infection in American kestrels. Kestrels with the HPAIV showed neurologic signs and eye disorders. The HPAIV replicated in systemic tissues of kestrels, and was orally shed. The HPAIV was recovered from feather calamus of kestrels.

The potential role of scavengers in spreading African swine fever among wild boar.

Understanding the transmission patterns of African swine fever (ASF) among wild boar (Sus scrofa) is an issue of major interest, especially in the wake of the current ASF epidemic. Given the high stability of ASF-virus, there is concern about scavengers spreading infectious carcass material in the environment. Here, we describe scavenging activities on 32 wild boar carcasses in their natural habitat in Germany. Using digital cameras, we detected 22 vertebrates at the study sites, thereof two mammal and three bird species scavenging. The most frequently detected species was the raccoon dog Nyctereutes procyonoides (44% of all visits). Raccoon dogs, red foxes (Vulpes vulpes), and buzzards (Buteo buteo) scavenged in the warm and the cold season, while ravens (Corvus corax) and white-tailed eagles (Haliaeetus albicilla) scavenged only in the cold season. In summer, however, insects removed most of the carcass biomass. Although most of the material was consumed on the spot, foxes, raccoon dogs and ravens left the study sites in rare cases with a small piece of meat in their mouths or beaks. We conclude that scavengers represent a minor risk factor for spreading ASF, but may contribute to reducing local virus persistence by metabolizing infected carcasses.

Detection of avian metapneumovirus subtype A from wild birds in the State of São Paulo, Brazil / Detecção de metapneumovirus aviário subtipo A em aves silvestres no estado de São Paulo, Brasil

The present study investigated the circulation of avian metapneumovirus (aMPV) in wild birds in Brazil. To do so, 131 samples from 366 oropharyngeal or cloacal swabs collected from 18 species of birds were tested individually or in pools by RT-PCR. Samples detected by RT-PCR were selected for DNA sequencing. Thirteen (9.9%) samples were detected by the RT-PCR targeting the N gene and four out of 13 samples were sequenced. Sequencing results showed a high identity with the aMPV subtype A. Our results confirm the circulation of the aMPV subtype A in wild birds in Brazil even five years after its last detection.(AU)

O presente estudo investigou a circulação de metapneumovírus aviário em aves silvestres no Brasil. Para tanto, 131 amostras de 366 suabes orofaringeanos ou cloacais coletados de 18 espécies de aves foram testadas individualmente ou na forma de pools por RT-PCR. As amostras detectadas por RT-PCR foram selecionadas para sequenciamento. Treze (9,9%) das amostras foram detectadas por RT-PCR tendo o gene N como alvo; destas, quatro foram sequenciadas com sucesso. Resultados do sequenciamento mostraram alta identidade com o aMPV de subtipo A. Nossos resultados confirmam a circulação de aMPV subtipo A em aves silvestres no Brasil mesmo cinco anos após sua última detecção.(AU)

Detection and Characterization of an Avipoxvirus in a Common Buzzard ( Buteo buteo) in Italy Using a Multiple Gene Approach.

Poxvirus infections have been reported in domestic, captive, and wild avian hosts including many raptor species. A wild Common Buzzard ( Buteo buteo) admitted to a wildlife veterinary clinic in Sardinia, Italy, showed multiple, wart-like proliferative cutaneous lesions on both legs. Histologically, there was ballooning degeneration and large intracytoplasmic inclusion bodies consistent with avipoxvirus (APV) infection. Diagnosis was confirmed by PCR detecting APV genes: P4b (locus fpv167), P35 (locus fpv140), and partial DNA polymerase. Phylogenetic analyses were performed to compare the detected virus with a panel of selected APVs. Analyses of P4b and DNA polymerase assigned the virus to clade A (fowlpox virus), subclade A7, grouping with many other APVs previously isolated in birds of prey. Further research should highlight the diversity of avian pox viral strains circulating among Common Buzzards as well as the phylogenetic role of locus fpv140 (P35) in comparison with the more-conserved P4b and DNA polymerase genes.

West Nile virus host-vector-pathogen interactions in a colonial raptor.

BACKGROUND: Avian host species have different roles in the amplification and maintenance of West Nile virus (WNV), therefore identifying key taxa is vital in understanding WNV epidemics. Here, we present a comprehensive case study conducted on red-footed falcons, where host-vector, vector-virus and host-virus interactions were simultaneously studied to evaluate host species contribution to WNV circulation qualitatively. RESULTS: Mosquitoes were trapped inside red-footed falcon nest-boxes by a method originally developed for the capture of blackflies and midges. We showed that this approach is also efficient for trapping mosquitoes and that the number of trapped vectors is a function of host attraction. Brood size and nestling age had a positive effect on the number of attracted Culex pipiens individuals while the blood-feeding success rate of both dominant Culex species (Culex pipiens and Culex modestus) markedly decreased after the nestlings reached 14 days of age. Using RT-PCR, we showed that WNV was present in these mosquitoes with 4.2% (CI: 0.9-7.5%) prevalence. We did not detect WNV in any of the nestling blood samples. However, a relatively high seroprevalence (25.4% CI: 18.8-33.2%) was detected with an enzyme-linked immunoabsorbent assay (ELISA). Using the ELISA OD ratios as a proxy to antibody titers, we showed that older seropositive nestlings have lower antibody levels than their younger conspecifics and that hatching order negatively influences antibody levels in broods with seropositive nestlings. CONCLUSIONS: Red-footed falcons in the studied system are exposed to a local sylvatic WNV circulation, and the risk of infection is higher for younger nestlings. However, the lack of individuals with viremia and the high WNV seroprevalence, indicate that either host has a very short viremic period or that a large percentage of nestlings in the population receive maternal antibodies. This latter assumption is supported by the age and hatching order dependence of antibody levels found for seropositive nestlings. Considering the temporal pattern in mosquito feeding success, maternal immunity may be effective in protecting progeny against WNV infection despite the short antibody half-life measured in various other species. We conclude that red-footed falcons seem to have low WNV host competence and are unlikely to be effective virus reservoirs in the studied region.

Ljungan/Sebokele-like picornavirus in birds of prey, common kestrel (Falco tinnunculus) and red-footed falcon (F. vespertinus).

Ljungan and Sebokele viruses are thought to be rodent-borne (picorna)viruses in the genus Parechovirus. Using random amplification and next generation sequencing method a novel Ljungan/Sebokele-like picornavirus was identified in birds of prey. Viral RNA was detected in total of 1 (9%) of the 11 and 2 (28.6%) of the 7 faecal samples from common kestrels and red-footed falcons in Hungary, respectively. High faecal viral RNA load (4.77×106 genomic copies/ml) measured by qPCR. The complete genome of picornavirus strain falcon/HA18_080/2014/HUN (KY645497) is 7964-nucleotide (nt) long including a 867-nt 5'end and a 101-nt 3'end (excluding the poly(A)-tail). Falcon/HA18_080/2014/HUN has type-II IRES related to hunnivirus IRES, encodes a polyprotein lacking a leader protein, a VP0 maturation cleavage site and it predicted to encode three 2A proteins (2A1NPG↓P, 2A2NPG↓P and 2A3H-Box/NC), two of them end with 'ribosome-skipping' sites (DxExNPG↓P). Sequence analyses indicated that the ORF1 (6996nt) polyprotein (2331 amino acid - aa) of falcon/HA18_080/2014/HUN shares the highest aa identity, 59% and 57%, to the corresponding polyproteins of Ljungan and Sebokele viruses. This study reports the identification and complete genome characterization of a novel Ljungan/Sebokele-like picornavirus in faeces of birds of prey which suggests that the genetic diversity and the potential host species spectrum of Ljungan/Sebokele-like viruses in genus Parechovirus are wider than previously thought.

Divergent hepatitis E virus in birds of prey, common kestrel (Falco tinnunculus) and red-footed falcon (F. vespertinus), Hungary.

Hepatitis E virus (HEV), family Hepeviridae, has raised considerable public health concerns because of its zoonotic potential; however, the animal to animal transmissions and the natural chain of hepevirus infections in wildlife are less known. Using random amplification and next generation sequencing technology a novel HEV in birds of prey was serendipitously identified in Hungary. HEV RNA was detected in total of 2 (18%) of the 11 and 1 (14%) of the 7 faecal samples from common kestrels and red-footed falcons, respectively. High faecal viral load (2.03×10(8) genomic copies/ml) measured by qPCR. The complete genome of strain kestrel/MR22/2014/HUN (KU670940) HEV is 7033-nt long including a 35-nt 5'end and a 63-nt 3'end (excluding the poly(A)-tail). Sequence analyses indicated that the ORF1 (4920nt/639 aa), ORF2 (1989nt/662 aa) and ORF3 (360nt/119aa) proteins of kestrel/MR22/2014/HUN shared the highest identity (58.1%, 66.8% and 28.5%) to the corresponding proteins of ferret, rat and human genotype 4 Orthohepeviruses, respectively. Interestingly, the ORF3 protein is potentially initiated with leucine (L) using an alternate, non-AUG (UUG) start codon. This study reports the identification and complete genome characterization of a novel Orthohepevirus species related to mammalian HEVs in birds of prey. It is important to recognize all potential hosts, reservoirs and spreaders in nature and to reconstruct the phylogenetic history of hepeviruses.

Seroepidemiologic Survey of Potential Pathogens in Obligate and Facultative Scavenging Avian Species in California.

Throughout the world, populations of scavenger birds are declining rapidly with some populations already on the brink of extinction. Much of the current research into the factors contributing to these declines has focused on exposure to drug residues, lead, and other toxins. Despite increased monitoring of these declining populations, little is known about infectious diseases affecting scavenger bird species. To assess potential infectious disease risks to both obligate and facultative scavenger bird species, we performed a serosurvey for eleven potential pathogens in three species of scavenging birds in California: the California condor (Gymnogyps californianus), turkey vulture (Cathartes aura) and golden eagle (Aquila chrysaetos). California condors were seropositive for avian adenovirus, infectious bronchitis virus, Mycoplasma gallisepticum, avian paramyxovirus-2, West Nile virus (WNV) and Toxoplasma gondii. Golden eagles were seropositive for avian adenovirus, Chlamydophila psittaci and Toxoplasma gondii, and turkey vultures were seropositive for avian adenovirus, Chlamydophila psittaci, avian paramyxovirus-1, Toxoplasma gondii and WNV. Risk factor analyses indicated that rearing site and original release location were significantly associated with a positive serologic titer to WNV among free-flying condors. This study provides preliminary baseline data on infectious disease exposure in these populations for aiding in early disease detection and provides potentially critical information for conservation of the endangered California condor as it continues to expand its range and encounter new infectious disease threats.

Outbreaks of highly pathogenic avian influenza H5N1 clade 2.3.2.1c in hunting falcons and kept wild birds in Dubai implicate intercontinental virus spread.

Highly pathogenic avian influenza viruses (HPAIVs) of subtype H5N1 have continued to perpetuate with divergent genetic variants in poultry within Asia since 2003. Further dissemination of Asian-derived H5 HPAIVs to Europe, Africa and, most recently, to the North American continent has occurred. We report an outbreak of HPAIV H5N1 among falcons kept for hunting and other wild bird species bred as falcon prey in Dubai, United Arab Emirates, during the autumn of 2014. The causative agent was identified as avian influenza virus subtype H5N1, clade 2.3.2.1c, by genetic and phylogenetic analyses. High mortality in infected birds was in accordance with systemic pathomorphological and histological alterations in affected falcons. Genetic analysis showed the HPAIV H5N1 of clade 2.3.2.1c is a reassortant in which the PB2 segment was derived from an Asian-origin H9N2 virus lineage. The Dubai H5N1 viruses were closely related to contemporary H5N1 HPAIVs from Nigeria, Burkina-Faso, Romania and Bulgaria. Median-joining network analysis of 2.3.2.1c viruses revealed that the Dubai outbreak was an episode of a westward spread of these viruses on a larger scale from unidentified Asian sources. The incursion into Dubai, possibly via infected captive hunting falcons returning from hunting trips to central Asian countries, preceded outbreaks in Nigeria and other West African countries. The alarmingly enhanced geographical mobility of clade 2.3.2.1.c and clade 2.3.4.4 viruses may represent another wave of transcontinental dissemination of Asian-origin HPAIV H5 viruses, such as the outbreak at Qinghai Lake caused by clade 2.2 ('Qinghai' lineage) in 2005.

Genome analysis of a novel, highly divergent picornavirus from common kestrel (Falco tinnunculus): the first non-enteroviral picornavirus with type-I-like IRES.

Although the number of identified avian-borne picornaviruses (family Picornaviridae) is continuously increasing there remains several species-rich avian host groups, such as the order Falconiformes (with 290 bird species) from which picornaviruses have not been identified. This study reports the first complete genome of a novel, highly divergent picornavirus, named as Falcovirus A1 (KP230449), from the carnivorous bird, the common kestrel (Falco tinnunculus, order Falconiformes). Falcovirus A1 has the longest 3D(RdRp) genome region and distant phylogenetic relationship to the Hepatitis A virus 1 (Hepatovirus) and Avian encephalomyelitis virus 1 (Tremovirus). It has a type-I (enterovirus-like) IRES in the 5'UTR - identified for the first time among avian-borne picornaviruses suggesting that type-I IRES is not restricted only to enteroviruses and providing further evidence of mosaicism of this region among different picornavirus genera.

Phylogenetic analysis of Columbid herpesvirus-1 in rock pigeons, birds of prey and non-raptorial birds in Poland.

BACKGROUND: The identity of herpesviruses isolated in Europe from domestic pigeons (Columbid herpesvirus-1 - CoHV-1) as well as falcons and owls remains unknown. All these herpesviruses are antigenically and genetically related. The falcons and owls are thought to have become infected during the ingestion of pigeon meat thus suggesting the virus's capacity to infect a wide range of hosts. The aim of the conducted study was to detect the occurrence of CoHV-1 and estimating the similarities and differences in the DNA-dependent DNA polymerase gene of herpesviruses isolated from domestic pigeons, birds of prey and non-raptorial free-ranging birds in Poland. RESULTS: The study has shown the presence of CoHV-1 in 20.4% (18/88) in the examined birds. In case of one CoHV-1, infected Peregrine Falcon (Falco peregrinus), neurological signs were observed. Nucleotide sequencing of the DNA-dependent DNA polymerase gene, showed a high similarity among Polish strains (100%), independently from the species of the affected birds. Only one compared CoHV-1 strain - KP 21/23 originating from Germany showed a slightly lower similarity at a level of 99.1%. Further analysis has shown the identity of DNA-dependent DNA polymerase of CoHV-1 strains and other herpesviruses present in poultry as well as other birds ranged from 35.4 to 44.9%. Interestingly CoHV-1 infection was also confirmed for the first time in four non-raptorial birds. CONCLUSIONS: The current study has shown a high similarity of CoHV-1 strains and the possible transmission of herpesviruses between domestic rock pigeons and free-ranging birds including raptors and non-raptorial birds. Further studies focused on cloning and the analysis of the whole CoHV-1 genome which is needed to explain the role of the observed similarities and differences between field strains of columbid herpesviruses.

Spread of influenza virus A (H5N1) clade 2.3.2.1 to Bulgaria in common buzzards.

On March 15, 2010, a highly pathogenic avian influenza virus was isolated from the carcass of a common buzzard (Buteo buteo) in Bulgaria. Phylogenetic analyses of the virus showed a close genetic relationship with influenza virus A (H5N1) clade 2.3.2.1 viruses isolated from wild birds in the Tyva Republic and Mongolia during 2009-2010. Designated A/common buzzard/Bulgaria/38WB/2010, this strain was highly pathogenic in chickens but had low pathogenicity in mice and ferrets and no molecular markers of increased pathogenicity in mammals. The establishment of clade 2.3.2.1 highly pathogenic avian influenza viruses of the H5N1 subtype in wild birds in Europe would increase the likelihood of health threats to humans and poultry in the region.

Full genome sequence of a recombinant H5N1 influenza virus from a condor in southern China.

In this study, we report the first genomic information on an H5N1 avian influenza virus (AIV) isolated from a condor in Guangdong Province in southern China in 2003. Full genome sequencing and phylogenetic analyses show that it is a recombinant virus containing genome segments derived from the Eurasia and North America gene pools. This will be useful for analyses of the evolution of H5N1 AIV in southern China.

Avian influenza virus risk assessment in falconry.

BACKGROUND: There is a continuing threat of human infections with avian influenza viruses (AIV). In this regard falconers might be a potential risk group because they have close contact to their hunting birds (raptors such as falcons and hawks) as well as their avian prey such as gulls and ducks. Both (hunting birds and prey birds) seem to be highly susceptible to some AIV strains, especially H5N1. We therefore conducted a field study to investigate AIV infections in falconers, their falconry birds as well as prey birds. FINDINGS: During 2 hunting seasons (2006/2007 and 2007/2008) falconers took tracheal and cloacal swabs from 1080 prey birds that were captured by their falconry birds (n = 54) in Germany. AIV-RNA of subtypes H6, H9, or H13 was detected in swabs of 4.1% of gulls (n = 74) and 3.8% of ducks (n = 53) using RT-PCR. The remaining 953 sampled prey birds and all falconry birds were negative. Blood samples of the falconry birds tested negative for AIV specific antibodies. Serum samples from all 43 falconers reacted positive in influenza A virus-specific ELISA, but remained negative using microneutralisation test against subtypes H5 and H7 and haemagglutination inhibition test against subtypes H6, H9 and H13. CONCLUSION: Although we were able to detect AIV-RNA in samples from prey birds, the corresponding falconry birds and falconers did not become infected. Currently falconers do not seem to carry a high risk for getting infected with AIV through handling their falconry birds and their prey.

Fatal columbid herpesvirus-1 infections in three species of Australian birds of prey.

We document columbid herpesvirus-1 (CoHV-1) infection in two barking owls (Ninox connivens), a powerful owl (Ninox strenua) and an Australian hobby (Falco longipennis). Antemortem signs of infection were non-specific and the birds either died soon after they were identified as ill or were found dead unexpectedly. Gross postmortem findings were also not specific. Microscopically, marked to massive splenic and hepatic necrosis with the presence of eosinophilic inclusion bodies in remaining splenocytes and hepatocytes was found in all birds. Herpesvirus virions were identified in liver sections from one of the boobook owls by electron microscopy. Using CoHV-1-specific primers and polymerase chain reaction, CoHV-1 DNA was amplified from tissue samples from all birds. A comparison of these sequences to previously reported sequences of CoHV-1 found them to be identical or to vary by a single base pair. These findings increase the number of known species of birds of prey that are susceptible to CoHV-1 infection and indicate that rock pigeons (Columbia livia) should not be included in the diet of captive Australian birds of prey.

Livestock drugs and disease: the fatal combination behind breeding failure in endangered bearded vultures.

There is increasing concern about the impact of veterinary drugs and livestock pathogens as factors damaging wildlife health, especially of threatened avian scavengers feeding upon medicated livestock carcasses. We conducted a comprehensive study of failed eggs and dead nestlings in bearded vultures (Gypaetus barbatus) to attempt to elucidate the proximate causes of breeding failure behind the recent decline in productivity in the Spanish Pyrenees. We found high concentrations of multiple veterinary drugs, primarily fluoroquinolones, in most failed eggs and nestlings, associated with multiple internal organ damage and livestock pathogens causing disease, especially septicaemia by swine pathogens and infectious bursal disease. The combined impact of drugs and disease as stochastic factors may result in potentially devastating effects exacerbating an already high risk of extinction and should be considered in current conservation programs for bearded vultures and other scavenger species, especially in regards to dangerous veterinary drugs and highly pathogenic poultry viruses.

Isolation and molecular characterization of Newcastle disease viruses from raptors.

The present study was undertaken to detect and characterize Newcastle disease virus (NDV) in raptors. Cloacal and oropharyngeal swab samples were collected from 60 casualty raptors during January to March 2009 in Minnesota. Inoculation of all these samples (n=120) in 9-day-old embryonated hens' eggs resulted in isolation of haemagglutinating viruses in three samples from two bald eagles and one great horned owl. These three haemagglutinating viruses were confirmed as NDV by reverse transcription-polymerase chain reaction (RT-PCR) using fusion gene-specific primers, and were negative for avian influenza virus by RT-PCR. Further characterization revealed that all three possessed (112)GKQGRL(117) at the fusion gene cleavage site, indicating that they were lentogenic strains. Phylogenetic analysis revealed that all three isolates clustered with published class II genotype II NDVs. The nucleotide sequence homology of the three NDV isolates among themselves was 98.4 to 99.6% and the sequence homology with lentogenic strains from wild birds used for comparison varied between 94.5 and 100%. Detection of NDV strains from raptors merits further epidemiological studies to determine the prevalence of different NDV strains in raptors and their impact in relation to transmission to domestic poultry.

Prevalence of neutralizing antibodies to West Nile virus in Eleonora's Falcons in the Canary Islands.

Birds are the major amplifying host for West Nile virus (WNV), a flavivirus that may affect humans and transmitted by bloodsucking vectors. Eleonora's Falcons (Falco eleonorae) migrate to the Canary Islands annually from WNV-endemic regions. To investigate the possible role of Eleonora's Falcons in the circulation of WNV, we measured WNV-specific antibodies in 81 falcons captured in 2006. None of the nestlings but 14.8% of the adults had WNV-neutralizing antibodies. RT-PCR did not detect flaviviruses in nonculicine ectoparasites (n=231) of the falcons. These findings suggest that WNV infection did not occur locally, but rather on the wintering grounds or during migration.

Prevalence of West Nile virus in wild American Kestrels (Falco sparverius) of southern Quebec, Canada.

We assessed West Nile virus (WNV) antibody prevalence in American Kestrels (Falco sparverius) in southern Quebec by sampling 152 birds during the 2003-05 breeding seasons. We analyzed the dependence of antibody prevalence on age group, year, and gender; a significant relationship was detected only between age (adults vs. nestlings) and antibody prevalence. Furthermore, reproductive success did not appear to be linked with the prevalence of anti-WNV antibodies in this population.

Isolation and identification of a fowl adenovirus from wild Black Kites (Milvus migrans).

A fowl adenovirus was isolated from wild Black Kites (Milvus migrans) that died around Kashipur, Uttarakhand, India. This virus isolate produced cytopathic effects in chicken embryo liver (CEL) cells and reacted with fowl adenovirus 4 (FadV-4) antiserum in agar gel immunodiffusion and immunofluorescence tests. The virus isolate was neutralized by FadV-4 antiserum with a neutralization titer of 800. Electron microscopy of infected CEL cells showed the presence of hexagonal virion particles measuring in size about 80-100 nm. An amplicon of 1,223 base pairs was detected using polymerase chain reaction with primers designed to target the hexon gene of FadV-4.